Chimeric neuregulins and method of making and use thereof

ABSTRACT

Composition containing a chimeric neuregulin polypeptides and method of making such polypeptides are disclosed. The chimeric neuregulin comprises a first moiety of at least 10 amino acids, wherein the first moiety is derived from a first polypeptide; and a second moiety of at least 5 amino acids, wherein the second moiety is derived from a second polypeptide; wherein the first polypeptide is a neuregulin and the chimeric neuregulin exhibits an enhanced binding affinity to integrin, Erb 3, or Erb 4 comparing to that of the first neuregulin.

This application is a continuation application of U.S. patentapplication Ser. No. 13/627,555, filed on Sep. 26, 2012. The entirety ofthe aforementioned application is incorporated herein by reference.

FIELD

This application generally relates to compositions containing a chimericneuregulin and methods for prevention and treatment of neuronal andvascular damages with chimeric neuregulins.

BACKGROUND

Neuregulins are a family of multipotent growth factors that includesacetylcholine receptor inducing activities (ARIAs), growth factors,heregulins, and neu differentiation factors. Neuregulins' effects appearto be mediated by interaction with a class of tyrosine kinase receptorsrelated to the epidermal growth factor receptor. Neuregulins stimulatethe tyrosine phosphorylation of these receptors and the subsequentactivation of various signal transduction mechanisms. Neuregulins aresynthesized as transmembrane precursors consisting of either animmunoglobulin-like or cysteine-rich domain, and EGF-like domain atransmembrane domain and acytoplastic tail. Neuregulins have been knownto be involved in the survival and function of neuronal cells.Neuregulin is also expressed in vascular endothelial cells and itsreceptors are localized in the underlying smooth muscle cells.

SUMMARY

One aspect of the present application relates to a chimeric neuregulinpolypeptide having an integrin domain that binds to an integrin and anErb3/4 binding domain that binds to Erb3 and/or Erb4. The chimericneuregulin polypeptide comprises a neuregulin backbone derived from anative neuregulin polypeptide, and a donor fragment of at least oneamino acid, wherein the donor fragment (1) replaces a target fragment inthe native neuregulin polypeptide, wherein the donor fragment differsfrom the target fragment by at least one amino acid, or (2) is insertedinto an insertion site of the native neuregulin polypeptide, and whereinthe donor fragment forms at least a portion of the integrin bindingdomain and/or at least a portion of the Erb3/4 binding domain of thechimeric neuregulin polypeptide.

Another aspect of the present application relates to a chimericneuregulin, comprising: a first moiety of at least 10 amino acids,wherein the first moiety is derived from a first polypeptide; and asecond moiety of at least 5 amino acids, wherein the second moiety isderived from a second polypeptide; wherein the first polypeptide is aneuregulin and wherein the chimeric neuregulin exhibits an enhancedbinding affinity to integrin, Erb 3, or Erb 4 comparing to that of theneuregulin.

Another aspect of the present application relates to a pharmaceuticalcomposition comprising the chimeric neuregulin of the presentapplication and a pharmaceutically acceptable carrier.

Another aspect of the present application relates to a method forameliorating neuronal damage in a subject. The method comprisesadministering to the subject an effective amount of the pharmaceuticalcomposition of the present application.

Another aspect of the present invention relates to a method forpreventing or ameliorating secondary neuronal injury and inflammationfollowing traumatic brain injury (TBI). The method comprisesadministering into a subject in need of such treatment an effectiveamount of the pharmaceutical composition of the present application.

Another aspect of the present invention relates to a method forameliorating blood vessel damage caused by acute mechanical or chemicalassault in a subject. The method comprises administering to said subjectan effective amount of the pharmaceutical composition of the presentapplication.

Other aspects of the invention will become apparent to the skilledartisan by the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention of this application is better understood in conjunctionwith the following drawing, in which:

FIG. 1 is a protein sequence alignment of the integrin binding domainand Erb3/4 binding domain of various neuregulins.

FIG. 2 shows the consensus sequences of NRG1, NRG2, NRG3 and NRG4.

FIG. 3 shows the amino acid sequences of certain chimeric neuregulin.

FIGS. 4A-4F are pictures showing ErbB4 receptor expression in apoptoticand degenerating neurons. After ErbB4 immunohistochemistry, brainsections from rats MCAO were stained with Fluoro-Jade, a marker fordegenerating neurons. Many neurons in the cortex wereFluoro-Jade-positive (FIG. 4A). ErbB4 positive cells (FIG. 4B) wereco-localized in Fluoro-Jade-positive neurons (FIG. 4C). Similarly, TUNELstaining (FIG. 4D) and ErbB4 (FIG. 4E) were double-labeled (FIG. 4F) ina subpopulation of cells in the ipsilateral brain. Arrows indicateexamples of double-labeled cells. Scale bar is 40 μM in panels A-C and20 μM in FIGS. 4D-4F.

FIGS. 5A-5D are pictures showing ErbB4 expression inmacrophages/microglia but not astrocytes following MCAO. Sections fromthe ipsilateral hemisphere were double labeled with antibodies againstErbB4 (FIG. 5A) and GFAP (FIG. 5B). Cells in the peri-infarct regionsdid not show co-localization of ErbB4 and GFAP (FIG. 5C).Co-localization of ErbB4 and Mac-1/CD11b indicated that ErbB4 is foundin a subset of macrophages/microglia (FIG. 5D). (Double arrows indicateexamples of double labeled cells). Scale bar is 40 μM in panel A-C and20 μM in FIG. 5D.

FIGS. 6A-6D are pictures and graphs showing that NRG1β treatment reduceMCAO/reperfusion-induced brain infarction. Representative2,3,5-triphenyltetrazolium chloride (TTC) stained brain sections areshown from rats injected with vehicle (FIG. 6A; n=11), NRG-1β (FIG. 6B;n=7) or NRG-1α (FIG. 6C; n=3) before MCAO. Infarct volumes in brainsfrom vehicle and NRG-1 treated animals are shown in the graph (FIG. 6D).Values are presented as mean±SEM; * denotes significantly different fromrespective vehicle treated animals (P<0.01).

FIGS. 7A-7E are pictures showing that NRG1β suppressesMCAO/reperfusion-induced apoptotic damage in rat brain. Rats weresubjected to MCAO for 1.5 hours followed by reperfusion for 24 hours(representative views are shown for TUNEL labeling of rat brainsections; n=5 for each condition). TUNEL staining is found in the cortex(FIG. 7A) and striatum (FIG. 7B) following MCAO while no TUNEL stainingis seen in the cortex (FIG. 7C) and reduced levels are seen in thestriatum (FIG. 7D) in NRG1β-treated rats. The coronal brain image(˜bregma+1.2 mm) indicates the areas observed in the sections (FIG. 7E).Scale bar is 100 uM.

FIGS. 8A-8E are pictures and graphs showing that NRG1 treatment reducesMCAO/reperfusion-induced brain infarction. Representative TTC stainedcoronal brain sections are shown where rats were injected with vehicle(FIG. 8A) or NRG1 immediately after MCAO (FIG. 8B) and 4 hours afterreperfusion (FIG. 8C). Infarct volumes in brains from rats treated withvehicle (n=10) or NRG1 immediately after MCAO (R0; n=8), 4 hours afterreperfusion (R4; n=6) or 12 hours after reperfusion (R12; n=8) are showin the graph (FIG. 8D). Values are presented as mean±SD of all infarctvolumes for each experimental condition; * denotes significantlydifferent from respective vehicle treated animals (P<0.01). The timeline (FIG. 8E) illustrates the MCAO protocol and NRG1 injections.

FIG. 9 is a graph showing that NRG1 administration resulted in asignificant improvement in neurological outcome (* denotes P<0.01). NRG1was administered after MCAO and 4 hours of reperfusion. Neurologicalfunction was graded on a scale of 0-4 (normal score 0, maximal deficitscore 4). All animals were tested prior to surgery (controls; n=14) andafter treatment with NRG1 or vehicle. The NRG1 treated group (n=9)displayed a 33% improvement in neurological score compared with vehicletreated rats (n=5).

FIGS. 10A-10F are pictures showing that NRG1β prevents microglial andastrocytic activation following MCAO. Rats were subjected to MCAOfollowed by reperfusion for 24 hours (n=5 for each condition). NRG1β orvehicle was injected into the ECA. Sections were labeled forimmunohistochemistry with an antibody against ED-1. While no stainingwas seen in the contralateral side (FIG. 10A), ED-1 labeled cells arepresent in the ipsilateral hemisphere (FIG. 10B) following MCAO invehicle-treated animals. Few ED-1 positive cells are found in animalstreated with NRG-1β (FIG. 10C). Examples of ED-1 positive cells areindicated by the arrows. Scale bar is 50 μM. To assess astrocyticactivation, sections were labeled for immunohistochemistry with anantibody against GFAP. Compared to the contralateral control (FIG. 10D),heavy GFAP staining is found at the border or infarct (FIG. 10E)following MCAO in vehicle-treated animals. However, when rats weretreated with NRG1β, GFAP expression was dramatically reduced in theperi-infarct regions (FIG. 10F). * denotes infarct core or thecorresponding region in the contralateral control; # denotesnon-ischemic tissues or the corresponding region in the contralateralcontrol. Scale bar is 100 μM.

FIG. 11 is a composite of pictures and graphs showing that NRG1β reducesMCAO/reperfusion-induced IL-1β mRNA levels. Rats were treated with NRG1βor vehicle then subjected to MCAO. RNA was isolated and IL-1β mRNAexpression was measured by RT-PCR. The expression of IL-1 (panel a) andGAPDH (panel b) mRNA is shown (n=4 for each condition). Panel c showsthe average percentage of change±SEM in IL-1 mRNA levels fromNRG-β-treated rat compared to vehicle-treated controls afternormalization to GAPDH (* denotes P<0.05). I=ipsilateral hemisphere;C=contralateral hemisphere.

DETAILED DESCRIPTION

The present invention relates to chimeric neuregulins, methods of makingchimeric neuregulins, and methods and compositions for prevention andtreatment of neuronal and vascular damages with chimeric neuregulins.

DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

The term “neuregulin” as used herein, refers to a family of proteinsthat includes: neuregulin-1 (NRG1), neuregulin-2 (NRG2), neuregulin-3(NRG3), and neuregulin-4 (NRG4). Neuregulin-1, which has also beendescribed in the literature as acetylcholine receptor inducing activity(ARIA), glial growth factor (GGF), glial growth factor 2 (GGF2);heregulin and neu differentiation factor (NDF); sensory and motorneuron-derived factor (SMDF), HGL; HRG; HRG1; HRGA and MST131, furthercontains a number of isoforms that include type I NRG1, type II NRG1,type III NRG1, type IV NRG1, type V NRG1 and type VI NRG1. The term“NRG1,” as used herein, also includes all other NRG1 isoforms. NRG1isoforms are synthesized as transmembrane precursors consisting ofeither an immunoglobulin-like or cysteine-rich domain, an EGF-likedomain, a transmembrane domain and a cytoplasmic tail (Fischbach et al.,Annu Rev Neurosci, 1997. 20:429-58). NRG1 isoforms are generated fromone gene by alternative mRNA splicing, and most of them are synthesizedas part of a larger transmembrane precursor. The two major classes ofNRG1 include α and β isoforms. The NRG1β isoforms predominate in thenervous system, while a isoforms are prevalent in mesenchymal cells. Theβ isoforms are 100 to 1,000 fold more potent in stimulating AChRsynthesis in skeletal muscle and Schwann cell proliferation (Buonanno etal., Curr Opin Neurobiol, 2001. 11:287-96). The effects of NRG1 appearto be mediated by interaction with a class of tyrosine kinase receptorsrelated to the epidermal growth factor (EGF) receptor which includesErbB2, ErbB3 and ErbB4 (Burden et al., Neuron, 1997. 18:847-55). TheEGF-like domain of NRG1 appears to be sufficient for activation of ErbBreceptors and downstream signal transduction pathways (Holmes et al.,Science, 1992. 256:1205-1210). NRG1 stimulates the tyrosinephosphorylation of these receptors and the subsequent activation ofvarious signal transduction mechanisms including Map kinase, PI3 kinaseand CDK5 (Fu et al., Nat Neurosci, 2001. 4:374-81).

Neuregulin 2 (NRG2) is a novel member of the neuregulin family of growthand differentiation factors. Through interaction with the ErbB family ofreceptors, NRG2 induces the growth and differentiation of epithelial,neuronal, glial, and other types of cells. The gene consists of 12 exonsand the genomic structure is similar to that of neuregulin 1 (NRG1).NRG1 and NRG2 mediate distinct biological processes by acting atdifferent sites in tissues and eliciting different biological responsesin cells. The NRG2 gene is located close to the region for demyelinatingCharcot-Marie-Tooth disease locus, but is not responsible for thisdisease. Alternative transcripts encoding distinct isoforms have beendescribed (Chang, H., et al. Nature (1997) 387: pp. 509-12; Carraway, KL, et al. Nature (1997) 387: pp. 512-16).

Neuregulin 3 (NRG3) binds to the extracellular domain of the ERBB4receptor tyrosine kinase but not to the related family members ERBB2 orERBB3. NRG3 binding stimulates tyrosine phosphorylation of ERBB4.Variants of the NRG3 gene have been linked to a susceptibility toschizophrenia (Zhang D, et al. Proc. Natl. Acad. Sci. U.S.A. (1997) 94:pp. 9562-7; Chen, P L, et al. Am. J. Hum. Genet. (2009) 84: pp. 21-34).

Neuregulin 4 (NRG4) activates type-1 growth factor receptors (EGFR) toinitiating cell-to-cell signaling through tyrosine phosphorylation. Lossof expression of NRG4 is frequently seen in advanced bladder cancerwhile increased NRG4 expression correlates to better survival (Harari,D., et al. Oncogene (1999) 18: pp. 2681-9); (Memon, A A., et al., Br. J.Cancer (2004) 91: pp. 2034-41)).

Examples of human neuregulin sequences include, but are not limited to,those listed under GenBank Accession Nos: ADN85612.1, AAF28851.1,AAF28850.1, AAF28849.1, AAF28848.1, ABR13844.1, ABR13843.1, EAW63417.1,EAW63416.1, EAW63415.1, AAI50610.1, ABQ53540.1, DAA00042.1, DAA00041.1,EAW62087.1, EAW62086.1, EAW62085.1, EAW62084.1, EAW62082.1, EAW62081.1,ADK90032.1, ADK90031.1, ADK90030.1, ADK90029.1, ADK90026.1, ADK90024.1,ADK90022.1, AAF28853.1, AAF28852.1, BAG70289.1, BAG70145.1, ABR13842.1,ABG77979.1, DAA00045.1, EAW63419.1, EAW63412.1, EAW63410.1, EAW63409.1,EAW63408.1, ADN85613.1, AAI36812.1, ABQ53539.1, DAA00048.1, DAA00047.1,EAW62083.1, EAW62078.1, ADK90028.1, ADK90027.1, ADK90025.1, ADK90023.1,ADK90021.1, ADK90020.1, AAH73871.1, ABY66350.1, EAW99228.1, EAW99227.1,EAW62080.1, EAW62079.1, EAW63418.1, EAW63414.1, EAW63413.1, EAW63411.1,EAW63407.1, EAW80374.1, AAH64587.1, AAH06492.1, AAO49724.1, AAH07675.1,AAP36053.1, AAH17568.1, CAI15622.1, CAH73645.1, CAH70641.1, CAI17213.1,CAH71050.1, AAM71141.1, AAM71140.1, AAM71139.1, AAM71138.1, AAM71137.1,AAM71136.1, AAM71135.1, AAM71134.1, AAM71133.1, CAI22410.1, ABQ53543.1,ABQ53542.1, ABQ53541.1, DAA00046.1, DAA00044.1, DAA00043.1, DAA00040.1,CAL35830.1, CAL35831.1, CAL35829.1, BAD97155.1, NP_(—)001159445.1,NP_(—)001159444.1, NP_(—)001010848.2, NP_(—)004874.1, NP_(—)053585.1,NP_(—)001171864.1, NP_(—)053586.1, NP_(—)053584.1, Q02297.3,NP_(—)001153467.1, NP_(—)039258.1, NP_(—)039251.2, NP_(—)001153473.1,NP_(—)001153471.1, NP_(—)039250.2, NP_(—)001153476.1, NP_(—)612640.1,NP_(—)039254.1, NP_(—)001153468.1, NP_(—)039256.2, NP_(—)001153480.1,014511.1, Q9H013.3, Q7RTV8, NP_(—)039252.2, NP_(—)001153479.1,NP_(—)001153474.1, NP_(—)001153477.1, NP_(—)004486.2, NP_(—)039253.1,P56975.1, AAA19954.1, AAA19953.1, AAA19951.1, AAA19950.1, AAB59358.1;AAB59622.1; AAI03985.2, AAI03985.2, AAI14335.2, AAI03984.2, Q8WWG1.1,AAA58640.1, AAA58639.1, AAA58638.1, AAC51756.1, AAY17216.1, 1910316A,ABY70644.1, ABY66349.1, ABY66348.1, ABC69293.1, AAA19952.1; AAA19955.1;AAA58641.1; AAB59358.1; AAB59622.1; AAC41764.1; AAH73871.1; AAI14334.2;AAI36812.1; AAO49724.1; AAP36053.1; ABC69293.1; ABQ53539.1; ABQ53540.1;ABR13843.1; ABR18344.1; ABY66350.1; BAA23417.1; BAD97155.1; BAF83419.1;BAF82616.1; BAG54044.1; BAG53780.1; BAG59183.1; BAH11473.1; BAH11479.1;BAH12729.1; CAD98015.1; CAG29284.1; CAH18333.1

The term “integrin binding domain of a NRG” or “integrin binding domainof an NRG consensus sequence,” as used herein, refers to the amino acidsequence in a native neuregulin that is located between and includingthe first and the third cyctienes of the NRG consensus sequence shown inFIGS. 1 and 2.

The term “Erb3/4 binding domain of a NRG” or “Erb3/4 binding domain ofan NRG consensus sequence,” as used herein, refers to the amino acidsequence in a native neuregulin that is located between and includingthe last two cyctienes residues (i.e., the fifth and sixth cyctienes) ofthe NRG consensus sequence shown in FIGS. 1 and 2.

The term “non-neuregulin polypeptide,” as used herein, refers to apolypeptide that is not a member of the neuregulin family.

The term “polypeptide” refers to a polymer in which the monomers areamino acid residues which are joined together through amide bonds. Theterms “polypeptide” or “protein” as used herein are intended toencompass any amino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those which are recombinantlyor synthetically produced. The term “fragment,” in reference to apolypeptide, refers to a portion (that is, a subsequence) of apolypeptide. A fragment contains at least three amino acids residues. Insome embodiments, the fragment contains at least 5 or 10 amino acidsresidues. Orientation within a polypeptide is generally recited in anN-terminal to C-terminal direction, defined by the orientation of theamino and carboxy moieties of individual amino acids. Polypeptides aretranslated from the N or amino-terminus towards the C orcarboxy-terminus.

The term “derived from” refers to the origin of a fragment or apolypeptide. A fragment or polypeptide is deemed to be “derived from” aprotein if the amino acid sequence of the fragment or the polypeptide isidentical to the amino acid sequence of the protein, or a portion orportions of the amino acid sequence of the protein. For example, apolypeptide with a sequence of HHHHKKK is deemed to be derived from apolypeptide with a sequence of HHHHCCCKKK.

“Solubility” is a measure the amount of a substance, in the context ofthis disclosure, a polypeptide, that will dissolve in a given amount ofanother substance, usually a liquid. Thus, an increase in solubility isan increase in the amount of a the polypeptide that remains withoutaggregating or separating from the substance (e.g., liquid) in which itis dissolved.

When referring to a polypeptide, “stability is a measure of thepolypeptide's resistance to degradation. Thus, an increase in stabilityreflects an increase in the ability of the polypeptide to withstanddegradation, for example, measured as an increased half-life in vivo, oran increased shelf life in vitro.

The term “enhanced binding affinity” is a relative term that refers toan binding affinity that is at least 50%, 100% or 200% above a baselinebinding affinity. For example, if the binding affinity between anintegrin and a chimeric neuregulin is more than 150% of the bindingaffinity between the same integrin and a native neuregulin (i.e., anover 50% increase in binding affinity), the chimeric neuregulin has an“enhanced binding affinity” to the integrin with regard to the nativeintegrin.

The term “reduces” is a relative term, such that an agent reduces aresponse or condition if the response or condition is quantitativelydiminished following administration of the agent, or if it is diminishedfollowing administration of the agent, as compared to a reference agent.Similarly, the term “prevents” does not necessarily mean that an agentcompletely eliminates the response or condition, so long as at least onecharacteristic of the response or condition is eliminated. Thus, acomposition that reduces or prevents an infection or a response, such asa pathological response, can, but does not necessarily completelyeliminate such an infection or response, so long as the infection orresponse is measurably diminished, for example, by at least about 50%,such as by at least about 70%, or about 80%, or even by about 90% of(that is to 10% or less than) the infection or response in the absenceof the agent, or in comparison to a reference agent.

The term “subject” refers to an individual. Preferably, the subject is amammal such as a primate, and, more preferably, a human. The term“subject” can include domesticated animals, such as cats, dogs, etc.,livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), andlaboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.).

The term “treatment” or “treating” refers to administration of acomposition to a subject with an undesired condition or at risk for thecondition. The condition can be any pathogenic disease, autoimmunedisease, cancer or inflammatory condition. The effect of theadministration of the composition to the subject can have the effect ofbut is not limited to reducing the symptoms of the condition, areduction in the severity of the condition, or the complete ablation ofthe condition.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed.

Chimeric Neuregulins

One aspect of the present invention relates to chimeric neuregulins thathave improved biological activities or pharmacokinetic characteristics.The term “chimeric neuregulin,” as used herein, refers to a polypeptidethat comprises a sequence derived from a neuregulin polypeptide and asequence derived from another polypeptide that can be a neuregulin or anon-neuregulin polypeptide.

In some embodiments, the chimeric neuregulin polypeptide has an integrindomain that binds to an integrin and an Erb3/4 binding domain that bindsto Erb3 and/or Erb4. The chimeric neuregulin polypeptide comprises aneuregulin backbone derived from a native neuregulin polypeptide, and adonor fragment of at least one amino acid, wherein the donor fragment(1) replaces a target fragment in the native neuregulin polypeptide,wherein the donor fragment differs from the target fragment by at leastone amino acid, or (2) is inserted into an insertion site of the nativeneuregulin polypeptide, and wherein the donor fragment forms at least aportion of the integrin binding domain and/or at least a portion of theErb3/4 binding domain of the chimeric neuregulin polypeptide. In oneembodiment, the chimeric neuregulin polypeptide has an enhanced bindingaffinity to integrin, Erb 3, or Erb 4, comparing to said nativeneuregulin peptide. In another embodiment, the donor fragment comprisesa polypeptide derived from another neuregulin that is different from thenative neuregulin. In another embodiment, the donor fragment comprises apolypeptide derived from a non-neuregulin. In another embodiment, thedonor fragment comprises polypeptides derived from two neuregulins thatare different from said native neuregulin. In another embodiment, theoriginal neuregulin polypeptide is NRG2 or a fragment of NRG2, and thedonor fragment comprises a polypeptide derived from NRG1β. In anotherembodiment, the native neuregulin polypeptide is NRG4 or a fragment ofNRG4, and the donor fragment comprises a polypeptide derived from NRG1,or a polypeptide derived from NRG2, or both a polypeptide derived fromNRG1 and a polypeptide derived from NRG2. In another embodiment, thechimeric neuregulin polypeptide further comprises a second donorfragment of at least one amino acid, wherein the second donor fragment(1) replaces a second target fragment in the native neuregulinpolypeptide, wherein the second donor fragment differs from the secondtarget fragment by at least one amino acid, or (2) is inserted into aninsertion site of the native neuregulin polypeptide.

In other embodiments, the chimeric neuregulin comprises a first moietyof at least 10 amino acids, wherein the first moiety is derived from afirst polypeptide; and a second moiety of at least 5 amino acids,wherein the second moiety is derived from a second polypeptide; whereinthe first polypeptide is a neuregulin and wherein the chimericneuregulin exhibits an enhanced binding affinity to integrin, Erb 3, orErb 4 comparing to that of the neuregulin. In one embodiment, the secondpolypeptide is a neuregulin that is different from the first neuregulin.In another embodiment, the second polypeptide is a non-neuregulinpolypeptide. In another embodiment, the chimeric neuregulin furthercomprises a third moiety derived from a third polypeptide. In a relevantembodiment, the third polypeptide is a neuregulin that is different fromthe native neuregulin. In another relevant embodiment, the secondpolypeptide is a second neuregulin and the third polypeptide is a thirdneuregulin, wherein the first neuregulin, second and third neuregulinsare different from each other. In another embodiment, the firstneuregulin is NRG2, and the second polypeptide is NRG1β. In anotherembodiment, the first neuregulin is NRG4 and the second polypeptide isNRG1 or NRG2. In another embodiment, the first moiety comprises a NRGintegrin binding domain or a NRG Erb3/4 binding domain. In anotherembodiment, the first moiety comprises a NRG integrin binding domain,and the second moiety comprises a NRG Erb3/4 binding domain. In anotherembodiment, the first moiety comprises a NRG Erb3/4 binding domain andthe second moiety comprises a NRG integrin binding domain.

In yet other embodiments, the chimeric neuregulin is a neuregulinpolypeptide (the original neuregulin) in which a stretch of at least 5,6, 7, 8 or 9 consecutive amino acid residues (the “original fragment”)is replaced with a stretch of consecutive amino acid residues (the“donor fragment”) from a donor polypeptide that is different from theoriginal neuregulin polypeptide. The original neuregulin can be a fulllength neuregulin or a fragment of a neuregulin. The donor polypeptidecan be another member of the neuregulin family or a non-neuregulinpolypeptide. The donor fragment may have a length that is the same as,or is different from, the length of the original fragment. A chimericneuregulin may contain more than one donor fragment. In one embodiment,one original fragment is replaced with two different donor fragments. Inother embodiment, two original fragments are replaced with two differentdonor fragments. In a preferred embodiment, the original fragmentcomprises an integrin binding domain of a NRG consensus sequence or anErb3/4 binding domain of a NRG consensus sequence.

In certain embodiments, the original neuregulin is NRG4 or a fragment ofNRG4 and the donor fragment(s) is derived from NRG1 and/or NRG2. Inother embodiments, the original neuregulin is NRG3 or a fragment of NRG3and the donor fragment(s) is derived from NRG1 and/or NRG2. In otherembodiments, the original neuregulin is NRG2 or a fragment of NRG2 andthe donor fragment(s) is derived from NRG1 and/or NRG2. In yet otherembodiments, the original neuregulin is NRG1 or a fragment of NRG1 andthe donor fragment(s) is derived from NRG1 and/or NRG2. In oneembodiment, the donor fragment is derived from NRG1β.

In certain embodiments, the chimeric neuregulin is a more effective Erb3 or Erb δ agonist than the original neuregulin polypeptide. In otherembodiments, the chimeric neuregulin has a higher affinity to Erb 3and/or Erb 4 than the original neuregulin. In other embodiments, thechimeric neuregulin has a higher affinity to integrin than the originalneuregulin. Selection of the original neuregulin, the original fragment,the donor polypeptide and the donor fragment can be determined based onthe sequence comparison and known Erb 3 and/or Erb 4 binding activity ofthe original neuregulin and the donor polypeptide.

For example, various neuregulins share the sequence homology shown inFIG. 1. Specifically, the neuregulin molecules all have a consensussequence that contains six cystiene residues spread in a region that isinvolved in binding to integrine and Erb3/4 molecules (see, e.g., FIGS.1 and 2). As shown in FIG. 1, NRG1, NRG2 and NRG3 all have either one ortwo positively charged amino acids (R, K or H) at the amino-end of thefirst cyctiene in the consensus sequence. NRGs with two positivelycharged amino acids (e.g., NRG2, NRG2.2, NRG 2.3 and NRG2.6) are able tobind integrins more effectively (i.e., having a higher affinity tointegrins). There integrins are up-regulated on inflamed tissue ortumors.

Further, the affinities of various NRGs to Erb3/4 have been reported asfollows:

NRG1β>NRG1α>NRG4 ( 1/10 of NRG1β)

NRG2>NRG3/NRG4 ( 1/10 the affinity of NRG2).

It thus appears that Erb3/4 interact with NRGs through the amino acidresidues between the last two cyctienes in the consensus sequence, aswell as the amino acid residues after the last cyctiene residue in theconsensus sequence.

Therefore, NRG2s have the highest affinities for integrins but NRG1αshave highest affinities for Erb 3/4. Therefore, a chimeric neuregulinwith the integrin binding domain of NRG2 and the Erb 3/4 binding domainof NRG1 would have similar affinities to integrin or Erb 3/4 whencompared to NRG2 or NRG 1β, respectively. However, such a chimericneuregulin would have a greater potency to bind to both integrin andErb3/4 when compared to NRG2 or NRG 113. For example, a NRG1/NRG2/NRG4chimeric neuregulin would have the ability to tightly bind neurotropicor vascular integrins while preserving the chimeric's ability to bindand inhibit ErbB3 and ErbB4 signaling. FIG. 3 shows certain embodimentsof chimeric neu e s (SEQ ID NOS:20-29).

As used herein, a “variant of a chimeric neuregulin” is a chimericneuregulin polypeptide that contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on thedesired bioactivity, secondary structure and hydropathic nature of thepolypeptide.

A variant of a chimeric neuregulin preferably exhibits at least about70%, more preferably at least about 80%, more preferably at least about90% and most preferably at least about 95% homology to the originalchimeric neuregulin polypeptide. Variants can be produced artificiallyusing site directed or random mutagenesis, or by recombination of two ormore preexisting variants. For example, a variant chimeric neuregulinpolypeptide can include 1, or 2, or 5 or 10, or 15, or up to about 50amino acid differences from the reference chimeric neuregulin.

Variants of chimeric neuregulin polypeptide also include polypeptidesthat are modified from the original chimeric neuregulin polypeptides byeither natural process, such as posttranslational processing, or bychemical modification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result frompost-translation natural processes or may be made by synthetic methods.Modifications include pegylation, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination.

Methods of Producing Chimeric Neuregulins

The chimeric neuregulins or variants thereof may be produced usingmethods well known in the art. In certain embodiments, the chimericneuregulins or variants thereof are produced by chemical synthesis.Briefly, a chimeric neuregulin may be synthesized by coupling thecarboxyl group or C-terminus of one amino acid to the amino group orN-terminus of another. Due to the possibility of unintended reactions,protecting groups are usually necessary. Chemical peptide synthesisstarts at the C-terminal end of the peptide and ends at the N-terminus.This is the opposite of protein biosynthesis, which starts at theN-terminal end.

In some embodiments, the chimeric neuregulines may be synthesized usingtraditional liquid- or solid-phase synthesis. Fmoc and t-Boc solid phasepeptide synthesis (SPPS) can be employed to grow the peptides fromcarboxy to amino-terminus. In certain embodiments, the last “amino acid”added to the reaction is PEGylated. This last amino acid is oftenreferred to as a carboxyl-PEG-amine, carboxyl-PEO-amine, oramine-PEG-acid, whereby the amine is blocked to protect against reactionand the acid is free to react with the amine group from the previouslyadded amino acid in the reaction. PEG (polyethylene glycol) and PEO(polyethylene oxide) are polymers composed of repeating subunits ofethylene glycol and ethylene oxide monomers. In one embodiment, aPEGylated GGF2/NRG2 chimeric (SEQ ID NO:28) would have the PEG moietyconnected to the histidine residue (H) at the amino-terminus of thepolypeptide. In one embodiment, the PEG moiety is 5 to 30 kDa in size.In another embodiment, the PEG moiety is 10 to 20 kDa in size.

In addition to using PEGylated end amino acid during synthesis, achimeric neuregulin may be PEGylated by PEGylation. PEGylation is theprocess of covalent attachment of polyethylene glycol polymer chains toanother molecule, normally a drug or therapeutic protein. PEGylation canbe achieved by incubation of a reactive derivative of PEG with thetarget chimeric neuregulin. The covalent attachment of PEG to a chimericneuregulin can “mask” the chimeric neuregulin from the host's immunesystem (reduced immunogenicity and antigenicity), increase thehydrodynamic size (size in solution) of the chimeric neuregulin whichprolongs its circulatory time by reducing renal clearance. PEGylationcan also provide water solubility to hydrophobic proteins.

The first step of the PEGylation is the suitable functionalization ofthe PEG polymer at one or both terminals. PEGs that are activated ateach terminus with the same reactive moiety are known as“homobifunctional”, whereas if the functional groups present aredifferent, then the PEG derivative is referred as “heterobifunctional”or “heterofunctional.” The chemically active or activated derivatives ofthe PEG polymer are prepared to attach the PEG to the desired molecule.

The overall PEGylation processes used to date for protein conjugationcan be broadly classified into two types, namely a solution phase batchprocess and an on-column fed-batch process. The simple and commonlyadopted batch process involves the mixing of reagents together in asuitable buffer solution, preferably at a temperature between 4 and 6°C., followed by the separation and purification of the desired productusing a suitable technique based on its physicochemical properties,including size exclusion chromatography (SEC), ion exchangechromatography (IEX), hydrophobic interaction chromatography (HIC) andmembranes or aqueous two phase systems.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, tyrosine. The N-terminal amino group and theC-terminal carboxylic acid can also be used as a site specific site byconjugation with aldehyde functional polymers.

In certain embodiments, the PEG derivatives are produced by reacting thePEG polymer with a group that is reactive with hydroxyl groups,typically anhydrides, acid chlorides, chloroformates and carbonates. Inother embodiments, more efficient functional groups such as aldehyde,esters, amides, etc. are made available for protein conjugation.

In certain embodiments, heterobifunctional PEGs are used forconjugation. These heterobifunctional PEGs are very useful in linkingtwo entities, where a hydrophilic, flexible and biocompatible spacer isneeded. Preferred end groups for heterobifunctional PEGs are maleimide,vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and NHSesters.

In other embodiments, the pegylation agents contain branched, Y shapedor comb shaped polymers that show reduced viscosity and lack of organaccumulation.

In other embodiments, chimeric neuregulins or variants thereof areproduced using recombinant DNA technologies. Procedures for theexpression and purification of recombinant proteins are well establishedin the art.

In order to express a chimeric neuregulin is a biological system, apolynucleotide that encodes the chimeric neuregulin is constructed. Incertain embodiments, the recombinant polynucleotide is codon optimizedfor expression in a selected prokaryotic or eukaryotic host cell, suchas a mammalian, plant or insect cell. To facilitate replication andexpression, the polynucleotide can be incorporated into a vector, suchas a prokaryotic or a eukaryotic expression vector. Although thepolynucleotide disclosed herein can be included in any one of a varietyof vectors (including, for example, bacterial plasmids; phage DNA;baculovirus; yeast plasmids; vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, pseudorabies, adenovirus, adeno-associated virus, retrovirusesand many others), most commonly the vector will be an expression vectorsuitable for generating polypeptide expression products. In anexpression vector, the polynucleotide encoding the neuregulin chimera istypically arranged in proximity and orientation to an appropriatetranscription control sequence (promoter, and optionally, one or moreenhancers) to direct mRNA synthesis. That is, the polynucleotidesequence of interest is operably linked to an appropriate transcriptioncontrol sequence. Examples of such promoters include: the immediateearly promoter of CMV, LTR or SV40 promoter, polyhedron promoter ofbaculovirus, E. coli lac or trp promoter, phage T7 and lambda P_(L)promoter, and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses. The expression vectortypically also contains a ribosome binding site for translationinitiation, and a transcription terminator. The vector optionallyincludes appropriate sequences for amplifying expression. In addition,the expression vectors optionally comprise one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells, such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or such as tetracycline or ampicillinresistance in E. coli.

The expression vector can also include additional expression elements,for example, to improve the efficiency of translation. These signals caninclude, e.g., an ATG initiation codon and adjacent sequences. In somecases, for example, a translation initiation codon and associatedsequence elements are inserted into the appropriate expression vectorsimultaneously with the polynucleotide sequence of interest (e.g., anative start codon). In such cases, additional translational controlsignals are not required. However, in cases where only a polypeptidecoding sequence, or a portion thereof, is inserted, exogenoustranslational control signals, including an ATG initiation codon isprovided for expression of the chimeric neuregulin sequence. Theinitiation codon is placed in the correct reading frame to ensuretranslation of the polynucleotide sequence of interest. Exogenoustranscriptional elements and initiation codons can be of variousorigins, both natural and synthetic. If desired, the efficiency ofexpression can be further increased by the inclusion of enhancersappropriate to the cell system in use. Expression vectors carrying thechimeric neuregulins can be introduced into host cells by any of avariety of well-known procedures, such as electroporation, liposomemediated transfection, calcium phosphate precipitation, infection,transfection and the like, depending on the selection of vectors andhost cells.

Host cells that contain chimeric neuregulins-encoding nucleic acids are,thus, also a feature of this disclosure. Favorable host cells includeprokaryotic (i.e., bacterial) host cells, such as E. coli, as well asnumerous eukaryotic host cells, including fungal (e.g., yeast, such asSaccharomyces cerevisiae and Picchia pastoris) cells, insect cells,plant cells, and mammalian cells (such as CHO cells). Recombinantchimeric neuregulin nucleic acids are introduced (e.g., transduced,transformed or transfected) into host cells, for example, via a vector,such as an expression vector. As described above, the vector is mosttypically a plasmid, but such vectors can also be, for example, a viralparticle, a phage, etc. Examples of appropriate expression hostsinclude: bacterial cells, such as E. coli, Streptomyces, and Salmonellatyphimurium; fungal cells, such as Saccharomyces cerevisiae, Pichiapastoris, and Neurospora crassa; insect cells such as Drosophila andSpodoptera frugiperda; mammalian cells such as 3T3, COS, CHO, BHK, HEK293 or Bowes melanoma; plant cells, including algae cells, etc.

The host cells can be cultured in conventional nutrient media modifiedas appropriate for activating promoters, selecting transformants, oramplifying the inserted polynucleotide sequences. The cultureconditions, such as temperature, pH and the like, are typically thosepreviously used with the host cell selected for expression, and will beapparent to those skilled in the art.

In bacterial systems, a number of expression vectors can be selecteddepending upon the use intended for the expressed product. For example,when large quantities of a polypeptide or fragments thereof are neededfor the production of antibodies, vectors which direct high levelexpression of fusion proteins that are readily purified are favorablyemployed. Such vectors include, but are not limited to, multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the coding sequence of interest, e.g., a polynucleotide of theinvention as described above, can be ligated into the vector in-framewith sequences for the amino-terminal translation initiating Methionineand the subsequent 7 residues of beta-galactosidase producing acatalytically active beta galactosidase fusion protein; pIN vectors (VanHeeke & Schuster (1989) J Biol Chem 264:5503-5509); pET vectors(Novagen, Madison Wis.), in which the amino-terminal methionine isligated in frame with a histidine tag, and the like.

Similarly, in yeast, such as Saccharomyces cerevisiae, a number ofvectors containing constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH can be used for production of thedesired expression products. In mammalian host cells, a numberexpression systems, including both plasmids and viral-based systems, canbe utilized.

A host cell is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, glycosylation, (as well as, e.g., acetylation,carboxylation, phosphorylation, lipidation and acylation).Post-translational processing for example, which cleaves a precursorform into a mature form of the protein (for example, by a furinprotease) is optionally performed in the context of the host cell.Different host cells such as 3T3, COS, CHO, HeLa, BHK, MDCK, 293, WI38,etc. have specific cellular machinery and characteristic mechanisms forsuch post-translational activities and can be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

For long-term, high-yield production of recombinant chimeric neuregulinpolypeptide, stable expression systems are typically used. For example,polynucleotides encoding a chimeric neuregulin polypeptides areintroduced into the host cell using expression vectors which containviral origins of replication or endogenous expression elements and aselectable marker gene. Following the introduction of the vector, cellsare allowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.For example, resistant groups or colonies of stably transformed cellscan be proliferated using tissue culture techniques appropriate to thecell type. Host cells transformed with a nucleic acid encoding achimeric neuregulin polypeptide are optionally cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture.

Following transduction of a suitable host cell line and growth of thehost cells to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. The secretedpolypeptide product is then recovered from the culture medium.Alternatively, cells can be harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Eukaryotic or microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents, or other methods, which are well know to thoseskilled in the art.

Expressed chimeric neuregulin polypeptides can be recovered and purifiedfrom recombinant cell cultures by any of a number of methods well knownin the art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein. Finally, high performance liquid chromatography(HPLC) can be employed in the final purification steps.

In certain examples, the nucleic acids are introduced into vectorssuitable for introduction and expression in prokaryotic cells, e.g., E.coli cells. For example, a nucleic acid including a polynucleotidesequence that encodes a F2GF1 chimeric RSV antigen can be introducedinto any of a variety of commercially available or proprietary vectors,such as the pET series of expression vectors (e.g., pET19b and pET21d).Expression of the coding sequence is inducible by IPTG, resulting inhigh levels of protein expression. The polynucleotide sequence encodingthe chimeric RSV antigen is transcribed under the phage T7 promoter.Alternate vectors, such as pURV22 that include a heat-inducible lambdapL promoter are also suitable.

The expression vector is introduced (e.g., by electroporation) into asuitable bacterial host. Numerous suitable strains of E. coli areavailable and can be selected by one of skill in the art (for example,the Rosetta and BL21 (DE3) strains have proven favorable for expressionof recombinant vectors containing polynucleotide sequences that encodeF2GF1 chimeric RSV antigens.

In another example, a polynucleotide sequence that encodes a chimericneuregulin is introduced into insect cells using a BaculovirusExpression Vector System (BEVS). Recombinant baculovirus capable ofinfecting insect cells can be generated using commercially availablevectors, kits and/or systems, such as the BD BaculoGold system from BDBioScience. Briefly, the polynucleotide sequence encoding the chimericneuregulin is inserted into the pAcSG2 transfer vector. Then, host cellsSF9 (Spodoptera frugiperda) are co-transfected by pAcSG2-chimer plasmidand BD BaculoGold, containing the linearized genomic DNA of thebaculovirus Autographa californica nuclear polyhedrosis virus (AcNPV).Following transfection, homologous recombination occurs between thepACSG2 plasmid and the Baculovirus genome to generate the recombinantvirus. In one example, the chimeric neuregulin is expressed under theregulatory control of the polyhedrin promoter (pH). Similar transfervectors can be produced using other promoters, such as the basic (Ba)and p10 promoters. Similarly, alternative insect cells can be employed,such as SF21 which is closely related to the SF9, and the High Five(Hi5) cell line derived from a cabbage looper, Trichoplusia ni.

Following transfection and induction of expression (according to theselected promoter and/or enhancers or other regulatory elements), theexpressed chimeric polypeptides are recovered (e.g., purified orenriched) and renatured to ensure folding into a biologically activeconformation.

In yet other embodiments, the chimeric neuregulins are expressed in vivoby a plasmid vector or a viral vector.

Methods for Treating Neuronal and Vascular Damages with ChimericNeuregulin

One aspect of the present application relates to a method forameliorating neuronal damage in a subject. The method includesadministering to the subject an effective amount of a pharmaceuticalcomposition comprising a chimeric neuregulin or a variant thereof,and/or an expression vector encoding a chimeric neuregulin or a variantthereof. The neuronal damage may be cause by an occlusive stroke, aneurotoxin, an acute CNS injury or traumatic brain injury (TBI).

Another aspect of the present application relates to a method forameliorating secondary neuronal injury and inflammation following TBI ina subject. The method includes administering to the subject an effectiveamount of a pharmaceutical composition comprising a chimeric neuregulinor a variant thereof, and/or an expression vector encoding a chimericneuregulin or a variant thereof.

Another aspect of the present application relates to a method fortreating or ameliorating symptoms of neurodegenerative disorders. Themethod includes administering to a subject suffering from aneurodegenerative disorder an effective amount of a chimeric neuregulinor a variant thereof, and/or an expression vector encoding a chimericneuregulin or a variant thereof. The term “neurodegenerative disorders”refer to diseases caused by progressive loss of structure or function ofneurons, including death of neurons. Examples of neurodegenerativedisorders include, but are not limited to, Alzheimer's disease,Parkinson's disease, Huntington's disease and amyotrophic lateralsclerosis.

While not to be bound by the theory, it is believed that administrationof the chimeric neuregulin or variant thereof results in themobilization and migration of endogenous neural stem cells (NSC) in vivoand can be used to stimulate adult neurogenesis.

The chimeric neuregulin may be administered by various routes, such asintrathecal administration, intravascular administration, intramuscularadministration, subcutaneous administration, intraperitonealadministration, oral administration and topical administration.Preferably, the neuregulin is administered intrathecally.

In certain embodiments, the chimeric neuregulin is administeredintrathecally in an amount sufficient to enhance migration of stem cellsfrom the ventricle into damaged areas of the brain. The chimericneuregulin may be administered via a shunt into the ventricle orsubventricular zone or administered into the cerebral spinal fluid byinjection at the lumbar region.

The use of shunts into the ventricle is well established practice in themedical community. Such shunts may be present for several day or weeks.While usually used to drain excess cerebral spinal fluid from theventricle in cases of excessive production or blockage of flow, suchshunts would be appropriate means for administration of chimericneuregulin over a period of several weeks. The care of the shunt wouldbe an ongoing responsibility of the medical team during the time thechimeric neuregulin is being administered to facilitate migration of thestem cells to areas of damage.

As used herein after “an effective amount of a chimeric neuregulin” is amount that is required to confer a prophylactic or therapeutic effect onthe treated subject, or an amount that is required to meliorate at leastone symptom of a target disease or disorder in the treated subject. Incertain embodiments, the chimeric neuregulin is used at an intrathecaldose range of 0.01 μg/kg body weight to 5 mg/kg body weight, 0.01 μg/kgbody weight to 500 μg/kg body weight, 0.01 μg/kg body weight to 50 μg/kgbody weight, 0.01 μg/kg body weight to 5 μg/kg body weight, 0.1 μg/kgbody weight to 5 mg/kg body weight, 0.1 μg/kg body weight to 500 μg/kgbody weight, 0.1 μg/kg body weight to 50 μg/kg body weight, 0.1 μg/kgbody weight to 5 μg/kg body weight, 1 μg/kg body weight to 5 mg/kg bodyweight, 1 μg/kg body weight to 500 μg/kg body weight, 1 μg/kg bodyweight to 50 μg/kg body weight, 10 μg/kg body weight to 5 mg/kg bodyweight, 10 μg/kg body weight to 500 μg/kg body weight and 10 μg/kg bodyweight to 50 μg/kg body weight.

In other embodiments, the chimeric neuregulin is used at anintra-vascular or intramuscular dose range of 0.01 μg/kg body weight to50 mg/kg body weight, 0.01 μg/kg body weight to 5 mg/kg body weight,0.01 μg/kg body weight to 500 μg/kg body weight, 0.01 μg/kg body weightto 50 μg/kg body weight, 0.01 μg/kg body weight to 5 μg/kg body weight,0.1 μg/kg body weight to 50 mg/kg body weight, 0.1 μg/kg body weight to5 mg/kg body weight, 0.1 μg/kg body weight to 500 μg/kg body weight, 0.1μg/kg body weight to 50 μg/kg body weight, 0.1 μg/kg body weight to 5μg/kg body weight, 1 μg/kg body weight to 50 mg/kg body weight, 1 μg/kgbody weight to 5 mg/kg body weight, 1 μg/kg body weight to 500 μg/kgbody weight, 1 μg/kg body weight to 50 μg/kg body weight, 10 μg/kg bodyweight to 50 mg/kg body weight, 10 μg/kg body weight to 5 mg/kg bodyweight, 10 μg/kg body weight to 500 μg/kg body weight and 10 μg/kg bodyweight to 50 μg/kg body weight, 100 μg/kg body weight to 50 mg/kg bodyweight, 100 μg/kg body weight to 5 mg/kg body weight, 100 μg/kg bodyweight to 500 μg/kg body weight and 1 mg/kg body weight to 50 mg/kg bodyweight. Administration can be a single bolus dose or multiple bolusdoses every day or every other day for a desired period of time (e.g., 5days, 10 days, 15 days, 20 days, 25 days, 30 days or longer). Thedesired treatment period is determined by the health care provider basedon the needs of each individual subject.

In certain embodiments, the chimeric neuregulin comprises an amino acidsequence selected from the group consisting of SEQ ID NOS:20-29. Incertain other embodiments, the chimeric neuregulin is a PEGylatedchimeric neuregulin. In other embodiments, the chimeric neuregulin isPEGylated at the N-terminal. In yet other embodiments, the PEG moiety inthe PEGylated chimeric neuregulin has a molecular weight of 10-20 Kd.

In some embodiments, the chimeric neuregulin is used in a method forameliorating neuronal damage of occlusive stroke. The method includesadministering to a subject suffering from an occlusive stroke aneffective amount of a chimeric neuregulin or a variant thereof, and/oran expression vector encoding a chimeric neuregulin or a variantthereof. In one embodiment, the chimeric neuregulin is administeredafter the onset of said occlusive stroke. In another embodiment, thechimeric neuregulin is administered within 24, 48 or 72 hours of theonset of said occlusive stroke. Preferably, the chimeric neuregulin isused in an amount that is effective in inhibiting inflammation in thesubject. In certain embodiments, the chimeric neuregulin is administeredin conjunction with a glutamate receptor inhibitor, a clot disruptingagent, such as t-PA, and/or other active agents. Examples of glutamatereceptor inhibitors include, but are not limited to, dizocilpin maleate(MK 801), R-2-amino-5-phosphonopentanoate (AP5),2-amino-7-phosphonoheptanoic acid (AP7),3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid (CPPene),PEAQX, Selfotel, Amantadine, Dextrallorphan, Dextromethorphan, Ethanol,Eticyclidine, Gacyclidine, Ibogaine, Ketamine, Magnesium, Memantine:Methoxetamine, Nitrous oxide, Phencyclidine, Rolicyclidine,Tenocyclidine, Methoxydine, Tiletamine, Xenon, Neramexane, Eliprodil,Etoxadrol, Dexoxadrol, WMS-2539, NEFA, Remacemide, Delucemine, 8A-PDHQ,Aptiganel (Cerestat, CNS-1102), HU-211, HU-210, Remacemide, Atomoxetine,and Rhynchophylline.

In certain other embodiments, the chimeric neuregulin is administeredafter the 72 hour window in an amount sufficient to enhance migration ofstem cells from the ventricle into damaged areas of the brain. In someembodiments, the chimeric neuregulin is administered daily or everyother day after the 72 hour window for 3-7, 3-14, 3-21 or 3-28 days.

The chimeric neuregulin may be administered by various routes, such asintravascular administration, intrathecal administration, intramuscularadministration, subcutaneous administration, intraperitonealadministration, oral administration and topical administration.Preferably, the chimeric neuregulin is administered intra-arterially.

In certain embodiments, the chimeric neuregulin is administeredintra-arterially in conjunction with a glutamate receptor inhibitor, aclot disrupting agent, and/or other active agents within 24, 48 or 72hours of the onset of said occlusive stroke to ameliorate acute damageand, then administered intrathecally after the 72 hour window for 3-7,3-14, 3-21 or 3-28 days in an amount sufficient to enhance migration ofstem cells from the ventricle into damaged areas of the brain.

For purposes of regeneration of neuronal tissue, the administration ofchimeric neuregulin should commence after the initial inflammation dueto the assault has subsided. In certain embodiments, the administrationof chimeric neuregulin is started within 3, 4, 5, 6 or 7 days or within1, 2, 3 or 4 weeks or within 1, 2 or 3 months of the initial assault,and is administered daily or every other day or weekly for 1, 2, 3 4, 5or 6 weeks. Because it is necessary for the stem cells that havemigrated to be replenished in the ventricle, the intrathecaladministration of chimeric neuregulin should not be repeated at lessthan one week intervals. Longer intervals may be appropriate in order toallow greater replenishment of the stem cell supply in the ventricle.

In a preferred embodiment, the chimeric neuregulin is administered usingserum albumin as a carrier. In one embodiment, the chimeric neuregulinis administered arterially in 1% serum albumin. In certain embodiments,the chimeric neuregulin comprises a sequence selected from the groupconsisting of SEQ ID NOS:20-29. In certain other embodiments, thechimeric neuregulin is a PEGylated chimeric neuregulin.

The glutamate receptor inhibitor is used in an amount that is effectivein blocking the excitotoxic events of ischemia and decrease damage toneuronal tissue and injury arising from reperfusion. In certainembodiments, the chimeric neuregulin and/or glutamate receptor inhibitorare administered via the carotid artery. In other embodiments, thechimeric neuregulin and/or glutamate receptor inhibitor are administeredto a particular area via fluoroscopy guided catheter. Chimericneuregulin may also be administered intravenously in conjunction withreperfusion therapy following occlusion of coronary arteries. In otherembodiments, the chimeric neuregulin and/or glutamate receptor inhibitorare administered intrathecally. The chimeric neuregulin and glutamatereceptor inhibitor may be administrated simultaneously or separately inany order.

In some other embodiments, the chimeric neuregulin is used in a methodfor ameliorating neuronal damage caused by exposure to neurotoxins, suchas organophosphates. The method includes administering to a subject inneed of such treatment an effective amount of a chimeric neuregulin or avariant thereof. In certain embodiments, the chimeric neuregulin orvariant thereof is administered in conjunction with the administrationof another active agent to ameliorate permanent damage from theneurotoxins. Examples of such active agents include, but are not limitedto, antidotes to neurotoxin and anticonvulsants.

Examples of neurotoxins include, but are not limited to,organophosphates, ion channel inhibitors, insects and animal venoms.Examples of antidotes to neurotoxin include, but are not limited to,atropine, prostigmine glutamate antagonists, oximes and benzodiazepies.

In certain embodiments, the chimeric neuregulin and/or other activeagent is given within 72 hours, preferably within 48 hours, morepreferably within 24 hours, from the initial exposure to the neurotoxin.In other embodiments, the chimeric neuregulin and/or other active agentare administered intrathecally or via the carotid artery. In otherembodiments, the chimeric neuregulin and/or other active agent areadministered to a particular area via fluoroscopy guided catheter.Chimeric neuregulin may also be administered intravenously orintramuscularly. The chimeric neuregulin and other active agent may beadministrated simultaneously or separately in any order.

In some other embodiments, the chimeric neuregulin is used in a methodfor ameliorating neuronal damage caused by neuronal cell deathassociated with CNS injury due to: Alzeimer's Disease, epilepsy,neonatal hypoxic ischemia, and seizures associated with traumatic braininjury, nerve agent exposure, ischemic stroke, hemorrhagic stroke,transient ischemic attacks, silent cerebral infarct, blunt force trauma.The method includes administering to a subject in need of such treatmentan effective amount of a chimeric neuregulin or a variant thereof.

Another aspect of the present invention relates a method for preventingor ameliorating secondary neuronal injury and inflammation followingtraumatic brain injury (TBI). The method comprises the step ofadministering into a subject in need of such treatment an effectiveamount of a chimeric neuregulin or a variant thereof, or an expressionvector encoding a chimeric neuregulin or a variant thereof.

In certain embodiments, the chimeric neuregulin and/or other activeagent is given within 72 hours, preferably within 48 hours, morepreferably within 24 hours of TBI. In other embodiments, the chimericneuregulin and/or other active agent are administered intrathecally orvia the carotid artery. In other embodiments, the chimeric neuregulinand/or other active agent are administered to a particular area viafluoroscopy guided catheter. Chimeric neuregulin may also beadministered intravenously or intramuscularly. The chimeric neuregulinand other active agent may be administrated simultaneously or separatelyin any order.

In a preferred embodiment, the chimeric neuregulin is administered usingserum albumin as a carrier. In one embodiment, the chimeric neuregulinis administered arterially in 1% serum albumin. In certain embodiments,the chimeric neuregulin comprises a sequence selected from the groupconsisting of SEQ ID NOS:20-29. In another embodiment, the chimericneuregulin is a PEGylated chimeric neuregulin.

Another aspect of instant invention relates to a method for amelioratingblood vessel damage caused by acute mechanical or chemical assault. Themethod comprises the step of administering to a subject in need of suchtreatment an effective amount of a chimeric neuregulin or a variantthereof, or an expression vector encoding a chimeric neuregulin or avariant thereof. While not to be bound by the theory, it is believedthat the chimeric neuregulin or variant thereof prevents damages arisingfrom responses to invasive procedures by inhibiting mitogen-stimulatedVSMC proliferation and migration.

In certain embodiments, the acute vascular conditions are caused byphysical assault to the blood vessels, such as placement of a balloon orstent in the artery, diagnostic cardiac catheterization, and cardiacsurgery, especially surgery on the heart valves. The neuregulintreatment provides protection from permanent damage to blood vesselsfrom restenosis and artherosclerosis arising from such physical assault.In addition to administration of neuregulin by intravenous orintra-arterial route during or after the damaging occasion, a stent orcatheter for use in an invasive procedure may be coated with neuregulin.Other routes, such as intramuscular injection, may also be used.

In certain embodiments, the chimeric neuregulin is used at anintravascular dose range of 0.01 μg/kg body weight to 50 mg/kg bodyweight, 0.01 μg/kg body weight to 5 mg/kg body weight, 0.01 μg/kg bodyweight to 500 μg/kg body weight, 0.01 μg/kg body weight to 50 μg/kg bodyweight, 0.01 μg/kg body weight to 5 μg/kg body weight, 0.1 μg/kg bodyweight to 50 mg/kg body weight, 0.1 μg/kg body weight to 5 mg/kg bodyweight, 0.1 μg/kg body weight to 500 μg/kg body weight, 0.1 μg/kg bodyweight to 50 μg/kg body weight, 0.1 μg/kg body weight to 5 μg/kg bodyweight, 1 μg/kg body weight to 50 mg/kg body weight, 1 μg/kg body weightto 5 mg/kg body weight, 1 μg/kg body weight to 500 μg/kg body weight, 1μg/kg body weight to 50 μg/kg body weight, 10 μg/kg body weight to 50mg/kg body weight, 10 μg/kg body weight to 5 mg/kg body weight, 10 μg/kgbody weight to 500 μg/kg body weight and 10 μg/kg body weight to 50μg/kg body weight, 100 μg/kg body weight to 50 mg/kg body weight, 100μg/kg body weight to 5 mg/kg body weight, 100 μg/kg body weight to 500μg/kg body weight and 1 mg/kg body weight to 50 mg/kg body weight.Administration can be a single bolus dose or multiple bolus doses everyday or every other day for a desired period of time (e.g., 5 days, 10days, 15 days, 20 days, 25 days, 30 days or longer). The desiredtreatment period is determined by the health care provider based on theneeds of each individual subject.

In a preferred embodiment, the chimeric neuregulin is administered usingserum albumin as a carrier. In one embodiment, the chimeric neuregulinis administered arterially in 1% serum albumin. In certain embodiments,the chimeric neuregulin comprises a sequence selected from the groupconsisting of SEQ ID NOS:20-29. In another embodiment, the chimericneuregulin is a PEGylated chimeric neuregulin. In other embodiments, thechimeric neuregulin is PEGylated at the N-terminal. In yet otherembodiments, the PEG moiety in the PEGylated chimeric neuregulin has amolecular weight of 10-20 Kd.

Pharmaceutical Compositions

Another aspect of the present invention relates to a pharmaceuticalcomposition for treating neurodegenrative disorder and preventing orameliorating neuronal damage caused by occlusive stroke, neurotoxin oracute assault on vascular and neuronal tissue. The pharmaceuticalcomposition contains a chimeric neuregulin or a variant thereof, and apharmaceutically acceptable carrier. In certain embodiments, theneuregulin comprises a sequence selected from the group consisting ofSEQ ID NOS:20-29.

As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, solubilizers, fillers,stabilizers, binders, absorbents, bases, buffering agents, lubricants,controlled release vehicles, diluents, emulsifying agents, humectants,lubricants, dispersion media, coatings, antibacterial or antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well-known in the art.See e.g., A.H. Kibbe Handbook of Pharmaceutical Excipients, 3rd ed.Pharmaceutical Press, London, UK (2000). Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary agentscan also be incorporated into the compositions.

In certain embodiments, the pharmaceutically acceptable carriercomprises serum albumin.

The pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intrathecal, intra-arterial,intravenous, intradermal, subcutaneous, oral, transdermal (topical) andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine; propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequited particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a neuregulin) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active, ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the pharmaceutical compositions areformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the pharmaceutical composition is formulated forsustained or controlled release of the active ingredient. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein includesphysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. In certain embodiments,single dosage contains 0.01 ug to 50 mg of a chimeric neuregulin.

Kits

The invention also encompasses kits for treating or preventing neuronaldamage caused by an occlusive stroke or exposure to neurotoxins, kitsfor treating neurodegenerative disorders, kits for treating orpreventing blood vessel damage caused by acute mechanical or chemicalassault, kits for treating or preventing acute CNS injuries, and kitsfor preventing or amelioratring secondary neuronal injury andinflammation following traumatic brain injury (TBI). The kits compriseone or more effective doses of a chimeric neuregulin, a variant of achimeric neuregulin, an expression vector encoding a chimeric neuregulinor a variant of a chimeric neuregulin, or combinations thereof alongwith a label or labeling with instructions on using the chimericneuregulin, the variant of chimeric neuregulin, or the expression vectoraccording to the methods of the invention. In certain embodiments, thekits can comprise components useful for carrying out the methods such asdevices for delivering the chimeric neuregulin, the variant of achimeric neuregulin, or the expression vector. In certain otherembodiments, the kits can further contain another active agent to beadministered in conjunction with the chimeric neuregulin, the variant ofchimeric neuregulin, or the expression vector.

EXAMPLES Example 1

Prevention of Post-Trauma Damage to Blood Vessels with Neuregulin-1

The effect of NRG1 on neointimal formation following balloon injury tothe carotid artery of the rat was examined. NRG1 (2.5 μg/kg) wasadministered by tail-vein injection prior to injury and every two daysfollowing injury. Two weeks after carotid artery injury, NRG1-treatedanimals demonstrated a 50% reduction in lesion size compared to controlsreceiving the vehicle. The effect of NRG1 on vascular smooth muscle cell(VSMC) function was studied. A7r5 rat VSMC cultures were pretreated withNRG1 for 24 hours, and then stimulated with platelet derived growthfactor (PDGF) for 48 hours. NRG1 significantly decreased both baselineand PDGF-stimulated VSMC proliferation in a dose-dependent manner. NRG1also blocked VSMC migration and prevented the downregulation of a-smoothmuscle actin by PDGF, indicating that it may prevent VSMC phenotypicreversion following injury. These findings demonstrate NRG1 as atherapeutic agent for the treatment of restenosis and atherosclerosis.

Experimental Injury, Harvest, and Tissue Preparation of Rat CarotidArteries

Male Sprague-Dawley rats (350-400 g) were balloon-injured using methodsas previously described in accordance with a protocol approved by theStanding Committee on Animals, Morehouse School of Medicine. Rats wereanesthetized with an intraperitoneal injection of xylazine (5 mg/kg bodyweight) and ketamine hydrochloride (90 mg/kg body weight). The leftcommon carotid artery was exposed by a 6-cm midline cervical incision.Proximal and distal blood flow was occluded by clamping. Polyethylene 10tubing was inserted retrogradely into the internal carotid artery andadvanced into the left common carotid artery. After gentle flushing ofthe artery with normal saline, the tubing was removed and a 2-French (F)Fogarty embolectomy balloon catheter was inserted. Balloon inflation to1.5 to 1.8 times the external diameter of the artery was achieved bycaliper measurement under stereomicroscopy. After holding the inflationfor 30 seconds, the catheter was removed. The uninjured right carotidartery was used as the control. Rats were treated with NRG1β or NRG-1α(EGF-like domain, R&D Systems, Minneapolis, Minn. dissolved in 1%BSA/PBS) by tail-vein administration at a dose of 2.5 ug/kg body weight,starting at day 0 before injury, and continuing for every 2 days for thenext 14 days. Control rats were treated with vehicle (1% BSA/PBS). Theanimals were weighed before the procedure and at sacrifice to evaluatethe possible adverse effects of NRG1. Vessels were harvested time points0 and 14 days for mRNA analysis or histology. Injured vessels werecompared with their contralateral controls.

Tissue Processing and Quantitative Histomorphometric Analysis

Animals were euthanized with CO₂ 14 days after injury. Carotid arterieswere washed with saline to clear blood, embedded in Tissue-Tek OCTmedium and frozen using liquid nitrogen. Carotid sections were cut witha cryostat into cross sections of 12 μm taken from the center and distalportion of the vessels, and stained with hematoxylin and eosin. Themedial thickness was determined by the area of the internal elasticlamina subtracted from the external elastic lamina. Morphometry wasperformed using at least six individual sections of each arterialsegment and used to determine the lesion size expressed as intima/mediaratio. The intimal and medial layer thicknesses were measured using acomputer-based image analyzing program (Image J, NIH).

A7r5 VSMC Cultures

A7r5 rat aortic vascular smooth muscle cells (VSMC) (ATCC CRL-1444) wereobtained from American Tissue Type Culture (Manassas, Va.) and grown inDulbecco's modified Eagle medium supplemented with glutamine, 10% fetalcalf serum (FCS), and 1% Penicillin/Streptomycin at 37° C. in ahumidified incubator with 5% CO₂. Cells were passaged weekly. Allstudies were performed on cells from passages 9-12.

Determination of VSMC Proliferation

VSMC were seeded at a density of 1×10³ cells in triplicate wells of a 96well plate. After 24 hours, cells were serum starved in DMEM/F-12(Gibco; Carlsbad, Calif.) containing 0.1% FCS (low serum medium; LSM) toinduce quiescence. After 24 hours of serum deprivation, cells werepretreated with 0-200 nM of NRG1α or NRG1β for 24 hours. Cells were thentreated with 10 ng/mL of PDGF-BB for 48 hours to stimulate VSMCproliferation. For direct measure of cell number, cells were countedusing a Coulter counter. VSMC cell proliferation and viability was alsomeasured using the CellTiter 96 AQueous Non Radioactive CellProliferation Assay (Promega; Madison, Wis.) according to themanufacturer's protocol. After incubation at 37° C. in humidified 5% CO₂for 1 hour, the absorbance was recorded at 490 nm using a plate reader.Measurement of DNA synthesis was performed using the BrDU CellProliferation Assay (Calbiochem, San Diego, Calif.) according to themanufacturer's protocol.

Cell Migration Assay

Neuro Probe 48-well microchemotaxis chambers (Costar, Corning Inc.) withPVP-free polycarbonate filter (8.0 μm pore size) were used to measureVSMC migration. Quiescent cells were trypsinized and resuspended in LSMwith or without NRG1 and incubated for 24 hours at 37° C. Cells werethen treated with PDGF which was added to the bottom well of the Boydenchamber and incubated for 48 hours at 37° C. Cells that migrated to thelower side of the filters were fixed and stained with the Diff Quickstaining kit (VWR Laboratory, West Chester, Pa.). The filters weremounted on glass slides and counted by light microscopy using ×100magnification.

Protein Purification and Western Analysis

Reactions were terminated by placing the cells on ice, aspirating themedium and adding ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 1 mMEDTA, 0.5% Triton X-100, 0.5% Nonidet P-40, 1 mM sodium orthovanadate, 1mM phenyl methanesulfonyl fluoride, pH 8.0) for 30 minutes at 4° C.Harvested lysates were denatured with loading buffer, resolved in SDS/5%polyacrylamide gels and transferred to poly vinylidene difluoride (PVDF)membranes (Millipore Corp., Bedford, Mass.). Membranes were be blockedwith 3% nonfat dry milk in phosphate buffered saline-0.5% Tween 20(PBST) and exposed to primary antibody, anti-smooth muscle alpha-actin(SMA) (Santa Cruz, Ca.) diluted in blocking buffer overnight at 4° C.After incubation, membranes were washed with PBST. After wash, membraneswere exposed to an alkaline phosphatase-conjugated anti-rabbit secondaryantibody for 1 hour. Membranes were subsequently washed with PBST,incubated with chemiluminescence reagents and exposed to x-ray film. ForERK1/2 phosphorylation, VSMC were pre-treated with NRG10 for 24 hoursand stimulated for 15 minutes with PDGF. Western blots were performedusing primary antibodies for phosphorylated and unphosphorylated formsof ERK1/2 (Cell Signaling, Danvers, Mass.) diluted 1:250 in blockingbuffer. Immunoblotting using an anti-tubulin antibody was used tonormalize protein levels in each sample.

Cell Viability Assay

Quantitative viability assessment was performed using 1% Calcein-AM(Molecular Probes, Eugene, Oreg.), a fluorescent membrane-integrity dye,diluted in HBSS according to the manufacturer's protocol. Qualitativeassessment of cell viability in treated cells was performed using thetrypan blue-exclusion assay. Non-viable cells were quantified visuallyusing a light microscopy.

Statistical Analysis

Each experiment was repeated a minimum of three times. Data areexpressed as the mean±standard deviation (SD). An unpaired Student'st-test and ANOVA were performed to make comparisons between groups. Avalue of p less than 0.05 was considered significant.

NRG1 Attenuated Neointima Formation after Rat Carotid Balloon Injury

Neointimal hyperplasia was histologically evident in the carotidarteries 14 days after balloon injury compared to uninjuredcontralateral controls. The neointima of the rats receiving intravenousadministration of NRG1 was significantly reduced compared toballoon-injured animals. Morphometric analysis showed that NRG1 reducedthe size of the lesion by ˜50% compared to vehicle-treated controlanimals. Treatment of animals with NRG1 showed no overt negative sideeffects and there was no significant difference in body weight observedamong the control and NRG1 treated rats.

NRG1 Inhibits Proliferation in VSMC

Serum-starved VSMC were pre-treated with NRG1 for 24 hours, thenstimulated with PDGF for an additional 48 hours. Stimulation of cellswith PDGF increased proliferation of VSMC 2-fold. Pre-treatment witheither NRG1β or NRG-1α resulted in a dose-dependent decrease in baselineand PDGF-stimulated proliferation as measured by MTS activity. Directcell counting using Coulter counter demonstrated that NRG1 reducedPDGF-stimulated VSMC proliferation, but not baseline cell numbers.Analysis of BrDU incorporation revealed a similar pattern to the Coultercounter demonstrating that NRG10 significantly inhibited PDGF-inducedproliferation, but did not alter baseline DNA synthesis.

Calcein-AM and trypan blue viability assays were carried out in cellspre-treated with NRG1 with or without PDGF. The calcein-AM assaydemonstrated that treatment of VSMC with NRG1 does not alter cellviability. These results were corroborated using the trypan-exclusionassay, which revealed that less than 1.0% of the cells took up the dye.

NRG1 Decreases VSMC Migration

The migration of VSMC was measured using a transwell migration assay.VSMC were pretreated with 100 nM NRG1α or NRG1β, and then stimulatedwith 10 ng/ml of PDGF-BB for 48 hours. The results show that NRG1 alonedoes not alter baseline VSMC migration. VSMC treated with PDGF displayeda 2-3 fold increase in migration. Both NRG-1α and NRG-1β decreasedPDGF-stimulated VSMC migration by 80% and 90%, respectively.

NRG1 Regulates Smooth Muscle α-Actin Expression

The mRNA and protein expression on SMA, and a marker for differentiatedand contractile VSMC, after NRG1 treatment was examined. Serum-starved,quiescent VSMC displayed SMA expression, which was reduced aftertreatment with PDGF. NRG1β alone did not alter SMA mRNA or proteinexpression, however, pre-treatment of PDGF-stimulated VSMC with NRG1βresulted in SMA expression that returned to near baseline levels.

NRG1 Inhibits PDGF-Induced Phosphorylation of ERK1/2

This study demonstrated that NRG 1 attenuates neointimal formation andvascular balloon injury. NRG1 reduced the size of the lesion by ˜50%compared to vehicle-treated control animals. This finding clearly showsthat NRG1 is useful in the prevention of vascular diseases such asrestenosis and atherosclerosis. The NRG1 blocks PDGF-inducedproliferation of VSMC in a dose-dependent manner. The inhibitory effectsof NRG1 on VSMC proliferation were confirmed by direct cell counting andmeasuring DNA synthesis by BrDU incorporation. An intriguing observationwas the difference in the effect of NRG1 on baseline VSMC proliferationusing the MTS-based assay compared to the other methods. In the cellcounting and BrDU approaches, PDGF increased VSMC proliferation wasblocked by NRG1, however, baseline VSMC numbers were not altered. Usingthe MTS-based assay, a 50% decrease in baseline MTS activity was seenafter NRG1 administration. Since the MTS assay measures metabolicactivity, it is possible that NRG1 may prevent PDGF-stimulatedproliferation by promoting VSMC differentiation, which could result in adecrease in metabolic activity and/or a reduction in the capability ofPDGF to stimulate VSMC proliferation. That this is due to apoptosisresulting from treatment is unlikely since there was no evidence ofincreased dead or non-viable cells after neuregulin treatment.

Example 2 Combination Therapy for Preventing Permanent Neuronal Damage

In the case of prevention of damage resulting from exposure toneurotoxins, such as organophosphates, or as a result of obstructivestroke, such as that caused by an infarct, studies were done studyingeffect on permanent middle cerebral artery occlusion (pMCAO) usingcombination therapy. Studies were done giving dizocilpin maleate (MK-801from Sigma), a glutamate receptor inhibitor which blocks the excitotoxicevents of ischemia in combination with neuregulin within a therapeuticwindow of about 13.5 hours in the rat to decrease permanent neuronaldamage. The therapeutic window in larger animals having a lowermetabolism would be in the range of 0 to 72 yours. In the case ofexpected exposure to neurotoxins, the neuregulin could be administeredin conjunction with antidotes. Other active agents which may be used inconjunction with neuregulin in the manner disclosed for use with MK-801are selfotel, aptiganel, magnesium, acetylcholine, GABA agonists(clomethiazole, diazepam and other benzodiazepines) and serotoninagonists.

In the case of damage arising from exposure to neurotoxins such asorganophosphate (OP) nerve agents, current post-exposure medicalcountermeasures against nerve agents (e.g. atropine, prostigmineglutamate antagonists, oximes (such as 20 pralidoxime chloride) andbenzodiazepines) are useful in preventing mortality, but are notsufficiently effective in protecting the CNS from seizures and permanentinjury. Therefore, new and more effective medical countermeasuresagainst OP nerve agents are needed to facilitate better treatment thatwill prevent extensive, permanent nerve damage in survivors. Otheragents that may be used to treat patients that have been exposed toneurotoxin include anticonvulsants.

In both instances of pMCAO and exposure to neurotoxins, neuregulin maybe administered concurrently with the other active agents to amelioratepermanent damage from infarct disintegrators or nerve agentcounteractants. In certain embodiments, neuregulin is given within a 72hour widow after the initial exposure to the causative agent or theonset of occlusion of the blood supply, more preferably within 24 hoursafter the causal event.

In both instances where the neuregulin is given as combination therapyto prevent cerebral neuronal damage the neuregulin is administered intothe carotid artery with an appropriate carrier. In animal studies, theneuregulin is administered in bovine serum albumin. In humans, apreferred carrier would be human serum to be administered within thefirst 72 hours, preferably within the first 24 hours, of the assault,whether chemical or physical. In the instance where the neuregulin is toprevent damage resulting from mechanical damage to a blood vessel, theneuregulin may be given intravenously in the usual carriers used forintravenous administration. Addressing the use of neuregulinsimultaneously with other agents, studies were done on rats that hadbeen subjected to left middle cerebral artery occlusion (MCAO).

Middle Cerebral Artery Occlusion

All surgical procedures were performed by sterile/aseptic techniques inaccordance with institutional guidelines. Adult male Sprague-Dawley ratsweighing 250-300 g were used for this study. Animals were subjected toleft MCA occlusion. Rats were anesthetized with a ketamine/xylazinesolution (10 mg/kg, i.p.). MCA occlusion was induced by the intraluminalsuture MCAO method as previously described (Belayev et al. 1996; Belayevet al. 1995). Briefly, the left common carotid artery (CCA) was exposedthrough a midline incision and was carefully dissected free fromsurrounding nerves and fascia. The occipital artery branches of theexternal carotid artery (ECA) were then isolated, and the occipitalartery and superior thyroid artery branches of the ECA were coagulated.The ECA was dissected further distally. The internal carotid artery(ICA) was isolated and carefully separated from the adjacent vagusnerve, and the pterygopalatine artery was ligated close to its originwith a 6-0 silk suture. Then, a 40 mm 3-0 surgical mono filament nylonsuture (Harvard Apparatus, Holliston, Mass.) was coated withpoly-L-lysine with its tip rounded by heating near a flame. The filamentwas inserted from the external carotid artery (ECA) into the internalcarotid artery (ICA) and then into the circle of Willis to occlude theorigin of the left middle cerebral artery. The suture was inserted 18 to20 mm from the bifurcation of the CCA to occlude the MCA. In thepermanent MCAO (pMCAO), the suture was left in place for 24 hours priorto sacrificing the animal. In the transient MCAO (tMCAO) model, thenylon suture was withdrawn 1.5 hours following ischemia and the braintissues were reperfused for 24 hours before sacrificing. To determinethe effects of NRG1 on ischemic stroke, rats were injectedintra-arterially with a single bolus 10 ul dose of vehicle (1% BSA inPBS) or NRG1β (10 μmol/L NRG1 (EGF-like domain, R&D Systems,Minneapolis, Minn.) in 1% BSA in PBS) through a Hamilton syringe.

NRG1 or vehicle was administered by bolus injection into the ICA throughECA immediately before MCAO. MK-801 (0.5 mg/kg) was either administeredIP immediately prior to NRG1 administration or co-administered IAsimultaneously with NRG1. All NRG1 and vehicle treatment studies wereperformed in a double-blinded manner. Core body temperature wasmonitored with a rectal probe and maintained at 37° C. with aHomeothermic Blanket Control Unit (Harvard Apparatus) during anesthesia.Neurological score was determined in a double blinded fashion using afive-point neurological evaluation scale (Menzies et al. 1992) in ratstreated with vehicle or NRG1 four hours after reperfusion. All animalswere tested prior to surgery (controls) and after treatment with NRG1 orvehicle. Neurological function was graded on a scale of 0-4 (normalscore 0, maximal deficit score 4). While intra-arterial injection intothe carotid artery was used, fluoroscopic guided catheter-based therapywherein the catheter is guided to the arteries which best access thedamaged tissue is appropriate.

Measurement of Infarct Formation

Twenty-four hours after reperfusion, the animals were killed and thebrain tissue was removed and sliced into 2.0 mm-thick sections. Brainslices were incubated in a 2% triphenyltetrazolium chloride (TTC)solution for 30 minutes at 3° C. and then transferred into a 4%formaldehyde solution for fixation. TTC, a colorless salt, is reduced toform an insoluble red formazan product in the presence of a functioningmitochondrial electron transport chain. Thus, the infarcted region lacksstaining and appears white, whereas the normal non-infarcted tissueappears red. Infarct area of four slices of 2 mm coronal sections ofeach brain was calculated in a blinded manner by capturing the imageswith a digital camera. Rats showing tremor and seizure (which rarelyoccurred in this study) were excluded from studies of brain infarctionto eliminate cerebral hemorrhage or brain trauma as potential variablesin this study. Infarct volumes were analyzed by ANOVA; P<0.05 wasregarded as significant.

The Effect of NRG1 on Neural Stem Cells (NSCs) Isolated from El1 MouseTelencephalon

The telencephalon of El1 mouse embryos were isolated and the dissociatedcells cultured as neurosphere cultures. Cultures were treated with theEGF-like domain of neuregulin-1β (NRG1). The EGF-like domain containsthe receptor binding portion of the molecule and has been shown todisplay all the known biological activities of the full-lengthneuregulins. The cells formed neurospheres and expressed nestin, anintermediate filament protein present in NSCs and RPs in the developingCNS. The cultures were examined to determine whether the addition ofNRG1 to cell suspensions obtained from El1 mouse cortical tissue wouldgenerate neurospheres in the absence of bFGF. After 7 days in culture,there was no significant difference in the total number or size ofneurospheres in the NRG1 treated group compared with the untreatedgroup. This result demonstrated that NRG1 alone, unlike bFGF, could notgenerate neurospheres. When bFGF-generated neurospheres were plated ontocoated coverslips in the presence of bFGF, cells continued to divide andmigrate out of the sphere to form a monolayer. Upon withdrawal of bFGF,migrating cells differentiated into cells expressing neuronal, astrocyteand oligodendrocyte markers. Neuronal cells were identified by labelingwith the anti-MAP2 antibody. Oligodendrocytes were identified with anantibody directed against O4 and astrocytes were identified with anantibody directed against GFAP. Morphologically, these MAP2-positivecells appeared neuronal and showed: (i) a spherical, ovoid, or pyramidalshaped soma; (ii) phase-bright appearance; (iii) branching processes(presumably dendrites) arising from the soma.

NRG1 Increases the Proliferation of MAP2-Positive Cells in NeurosphereCultures

The actions of NRG1 on βFGF-generated neurospheres were examined byplating neurospheres on coverslips as described above. Neurospheres werecultured in the absence or presence of 5 nM NRG1 for 5 days, and then,co-labeled with BrDU and MAP2 or GFAP antibodies. After 5 days oftreatment with 5 nM NRG1, a dramatic increase in the number of cellssurrounding the core of the neurosphere was observed in NRG1 treatedcultures as compared to control. A 44±3.3% increase in [3H]thymidineincorporation was seen in NRG1 treated cultures that paralleled theincrease in the total number of cells. More MAP2 and BrDU co-labeledcells were found both in the central core and peripheral area of NRG1treated neurospheres, but few double-labeled cells were seen in thecontrol. There was a 2.5-fold increase in MAP2 positive cells, but noincrease in MAP2-negative cells, suggesting that the majority of NRG 1treated cultures were neuronal.

To further characterize the effect of NRG1 on NSCs, neurospheres werecultured in the absence or presence of 1 or 5 nM NRG1, then co-labeledwith BrDU and MAP2 or GFAP antibodies. After 5 days, there was a 4-foldincrease in the number BrDU-labeled cells in the neurosphere outgrowtharea in 5 nM NRG1 treated group compared to control. A smaller, butsignificant increase was also observed with 1 nM NRG1 treatmentdemonstrating a dose-dependent response of cells to NRG1. Most of theBrDU positive cells co-labeled with the MAP2, but not the GFAP antibody.Therefore, the increased proliferation was specific for neuronal cellsand not in GFAP-positive astrocytes. The increase in number of MAP2positive cells that co-labeled with BrDU was parallel to the increase ofBrDU positive cells, suggesting that most of the cells proliferating inresponse to NRG1 were neuronal. In cultures maintained for 8 days afterwithdrawal of bFGF, virtually no cells showed BrDU incorporation incontrol cultures. By that time point, most cells had differentiated andlost the ability to proliferate. However, numerous BrDU-positive neuronswere present in the NRG1 treated group, suggesting that NRG1 prolongsthe proliferation of immature neuronal cells. Under our cultureconditions, few cells were labeled with GFAP and O4 in control cultures,or after treatment with NRG1, therefore the cells that labeled with BrDUalone were likely undifferentiated NSCs.

NRG1 Increases Proliferation Rather than Survival

The increased production of neurons could be altered by affecting (1)the proliferation of NRPs (2) the differentiation of NSCs into neurons,or (3) or by altering the survival of neuronal cells. Increasedproliferation might result in an increase in the total number of cellsas well as in the number of proliferating MAP2-positive cells; increaseddifferentiation might result in an increase in the number ofMAP2-positive cells within the same total population of cells; increasedsurvival might result in an increase in MAP2-positive cells, but notnecessarily cells co-labeled with BrDU or nestin. To determine whetherthe increase in the number of NRPs induced by NRG1 was due to theincrease of cell survival, we evaluated cell viability by using aViability. Assay Kit (Molecular probes). Results showed that the totalnumber of cells increased after 4 days in the cultures. The number oflive cells was greater in NRG1 treated group compared to control. Twiceas many live cells were present in the NRG1 group after 8 days. Therewas no difference in the number of dead cells in control or NRG1 treatedcultures at most time points. This result shows that NRG1 stimulatedproliferation rather than cell survival.

NRG1 Stimulates the Mobilization of NSCs in Adult Rat Brain In Vivo

SVZ cells were labeled by stereotaxically injecting DiI into the lateralventricle of adult rats. Twenty-four hours later, NRG 1 or vehicle wereinjected into the lateral ventricle and the animals were sacrificed 1day later. Intense labeling was visible in the cells lining theventricle (V) and in the choroids plexus (CP) after injection of DiI.When the vehicle was injected 24 after the DiI labeling, few cells hadmigrated out from the SVZ. However, after NRG1 administration, numerousNSCs had migrated from the SVZ, as far away as to the cerebral cortex.Similar results were seen when Fluorogold was injected into the lateralventricle. Preliminary results indicate that a subpopulation of thelabeled cells co-label with an antibody for NeuN, a neuronal marker.

The poor regenerative capacity of the sensory of the mammalian CNS hasled to investigations of different approaches to increase the functionof these structures after neurodegeneration or injury. One strategy torepair the injured CNS has been to replace the lost neurons withembryonic stem cell-derived neuronal stem cells (eNSCs). The use ofeNSCs has shown promise in the treatment of a variety of neurologicaldiseases and they have recently been shown to survive and differentiateinto glia and neurons after CNS transplantation. However, a number ofbiological and ethical issues have slowed this area of research. NSCshave been demonstrated in the adult brain and have been shown to havethe potential to differentiate into a variety of neuronal cell types.Therefore, another strategy has focused on maximizing the potential ofthis endogenous population of cells by stimulating their mobilization,proliferation, migration and differentiation in vivo following CNSinjury and degeneration. Understanding the technical and logisticconsiderations for employing adult NSCs is essential to optimizing andmaintaining cell survival before and after activation, as well as fortracking the fate of mobilized cells. It is now recognized that NSCstrategies will be effective only if the new cells have the sameabilities and characteristics as the original neurons. Theadministration of NRG1 into the cerebral spinal fluid or through a shuntinto the ventricle for repeated administration (intrathecaladministration) gives a method of encouraging production of stem cellsin the ventricle with migration of stem cells from the ventricle to thedamaged areas of the brain. Administration may be used with spinal tapor may be administered through a shunt into the ventricle. Appropriatecarriers include glucose, isotonic glucose and other carriers usuallyused for intrathecal administration. However, the neuregulin may beadministered into the cerebral spinal fluid at the lumbar region. Forarterial administration, the carrier may, advantageously, contain serumalbumin.

To obtain maximum benefit, the neuregulin will usually administer intothe carotid artery or by some other means such as fluoroscopy guidedcatheter-based means that will provide arterial access to the brain.

While it had previously been demonstrated that a single intra-arterialadministration of NRG1 prior to MCAO prevented neuronal death followingischemia and reperfusion, there was no indication that use at or aftertime of assault, whether mechanical (as with an infarct) or chemical,would be effective to ameliorate damage arising from the assault.

Neuregulin also has use, with similar dosage for intravenousadministration in conjunction with reperfusion therapy such asanticoagulant therapy to ameliorate damage to the artery. In suchinstances, the neuregulin may be administered in carriers such asglucose, saline, Ringer's lactate, etc.

Agents with other mechanisms of action that prevent or avoid formationof obstructive occlusion such as those which cause clots to dissolve canalso be used with neuregulin. In one embodiment, tissue plasminogenactivator (t-PA) is used in conjunction with neuregulin. At present, useof t-PA remains limited and must be administered within three hours ofthe observed ischemic event. However, t-PA patients are at high risk ofhemorrhagic transformation. Furthermore, t-PA causes inflammatoryresponses and reperfusion injury in the brain. The t-PA is administeredintravenously in saline or similar carriers. In all instances, theneuregulin is most effective if administered into the carotid artery ina carrier containing serum albumin (in the case of humans, human serumalbumin). The agents may be administered essentially simultaneously orthe neuregulin may be administered within the 0-72 hour time period,though it is preferred practice to administer the neuregulin within 24hours of administration of the t-PA.

While treatments cited above may be effective in limiting damage from apathology-causing event, the recovery of function can take place onlywith regeneration of neuronal tissue. The administration of neuregulininto the ventricular zone provides means for enhancing migration of stemcells which are formed in the ventricle to the site of neuronal damage.

Example 3 ErbB4 Receptors are Expressed Apoptotic and DegeneratingNeurons

After erB4 immunohistochemistry, brain sections harvested from ratssubjected to middle cerebral artery occlusion (MCAO) were stained withFluoro-Jade, a marker for degenerating neurons, and with antibodiesagainst ErbB4. As shown in FIGS. 4A-4F, many neurons in the cortex wereFluoro-Jade-positive (FIG. 4A). ErbB4 positive cells (FIG. 4B) wereco-localized in Fluoro-Jade-positive neurons (FIG. 4C). Similarly, TUNELstaining (FIG. 4D) and ErbB4 (FIG. 4E) were double-labeled (FIG. 4F) ina subpopulation of cells in the ipsilateral brain.

Example 4 ErbB4 Expression is Upregulated in Macrophages/Microglia butnot in Astrocytes Following MCAO

Sections from the ipsilateral hemisphere of rats subjected to MCAO weredouble labeled with antibodies against erbB4 (FIG. 5A) and glialfibrillary acidic protein (GFAP) (FIG. 5B). Cells in the peri-infarctregions did not show co-localization of erbB4 and GFAP (FIG. 5C).Co-localization of erbB4 and Mac-1/CD11b indicated that ErbB4 is foundin a subset of macrophages/microglia (FIG. 5D).

Example 5 NRG-1β Treatment Reduces MCAO/Reperfusion-Induced BrainInfarction

FIGS. 6A-6D show representative TTC stained brain sections from ratsinjected with vehicle (panel a; n=11), NRG1β (panel b; n=7) or NRG1α(panel c; n=3) before MCAO. NRG1β (2.5 μg/kg) or NRG1α (2.5 μg/kg) wasgiven by a single intra-arterial injection immediately before MCAO.Adult male Sprague-Dawley rats weighing 250-300 g were used for thisstudy. A total of 164 rats were used in this study. Rats wereanesthetized with a ketamine/xylazine solution (10 mg/kg, IP) andsubjected to left MCAO. MCAO was induced by the intraluminal suturemethod where the left common carotid artery (CCA) was exposed through amidline incision and was carefully dissected free from surroundingnerves and fascia. The occipital artery branches of the external carotidartery (ECA) were then isolated, and the occipital artery and superiorthyroid artery branches of the ECA were coagulated. The ECA wasdissected further distally. The internal carotid artery (ICA) wasisolated and carefully separated from the adjacent vagus nerve, and thepterygopalatine artery was ligated close to its origin with a 6-0 silksuture. Then, a 40 mm 3-0 surgical monofilament nylon suture (HarvardApparatus, Holliston, Mass.) was coated with poly-L-lysine with its tiprounded by heating near a flame. The filament was inserted from the ECAinto the ICA and then into the circle of Willis to occlude the origin ofthe left MCA. The suture was inserted 18 to 20 mm from the bifurcationof the CCA to occlude the MCA. After 1.5 hour of ischemia, the nylonsuture was withdrawn and the ischemic brain tissue was reperfused for 24hours before sacrificing. Core body temperature was monitored with arectal probe and maintained at 37° C. with a Homeothermic BlanketControl Unit (Harvard Apparatus) during anesthesia. To determine theeffects of NRG-1 on ischemic stroke, rat were injected intravascularlywith a single bolus 10 μl dose of vehicle (1% BSA in PBS) or NRG-1β(1-50 umol/L NRG-1 (EGF-like domain, R&D Systems, Minneapolis, Minn.)dissolved in 1% BSA/PBS) through a Hamilton syringe at a rate of 5μl/min. This resulted in the administration of 0.5-2.5 μg of NRG-1/kgbody weight. NRG-1 or vehicle was administered by bolus injection intothe ICA through ECA. Solutions were administered either before MCAO orimmediately following 1.5 hours of MCAO and either 0, 4 or 12 hours ofreperfusion. Animals were sacrificed 24 hours after reperfusion or after14 days for the long-term studies. Animals were killed 24 hours laterand the brains were sliced into 2 mm sections and stained with2,3,5-triphenyltetrazolium chloride (TTC). Infarct volumes in brainsfrom vehicle and NRG1 treated animals are shown in the graph (FIG. 6D).The data demonstrate that NRG1β treatment reducesMCAO/reperfusion-induced brain infarction.

Example 6 NRG-1β Suppresses MCAO/Reperfusion-Induced Apoptotic Damage inRat Brain

Rats were subjected to MCAO for 1.5 hours followed by reperfusion for 24hours. FIGS. 7A-7E show representative views of TUNEL labeling of ratbrain sections (n=5 for each condition). TUNEL assay was performed witha DeadEND Fluorometric TUNEL System (Promega, Madison, Wis.) accordingto the manufacturer's instructions. Slides were then washed with PBS andmounted with Vectashield Mounting Medium containing DAPI. All sectionswere examined by fluorescence microscopy in three random middle cerebralartery served areas in the inner border of the infarct in the ischemicfront-parietal cortex of each rat. In animals given vehicle orneuregulin-1, cortex and striatum were examined in three or more 20 lamsections per animal. TUNEL staining was found in the cortex (FIG. 7A)and striatum (FIG. 7B) following MCAO while no TUNEL staining was seenin the cortex (FIG. 7C) and reduced levels were seen in the striatum(FIG. 7D) in NRG1β-treated rats. The coronal brain image (˜bregma+1.2mm) indicates the areas observed in the sections (FIG. 7E).

Example 7 NRG-1 Treatment Reduces MCAO/Reperfusion-Induced BrainInfarction

FIGS. 8A-8E show] representative TTC stained coronal brain sections fromrats injected with vehicle (FIG. 8A) or NRG1 immediately after MCAO(FIG. 8B) and 4 hours after reperfusion (FIG. 8C). Infarct volumes inbrains from rats treated with vehicle (n=10) or NRG1 immediately afterMCAO (R0; n=8), 4 hours after reperfusion (R4; n=6) or 12 hours afterreperfusion (R12; n=8) are show in the graph (FIG. 8D). The time line(FIG. 8E) illustrates the MCAO protocol and NRG1 injections.

Example 8 NRG-1 Administration Resulted in a Significant Improvement inNeurological Outcome

NRG1 was administered after MCAO and 4 hours of reperfusion. As shown inFIG. 9, neurological function was graded on a scale of 0-4 (normal score0, maximal deficit score 4). All animals were tested prior to surgery(controls; n=14) and after treatment with NRG-1 or vehicle. The NRG1treated group (n=9) displayed a 33% improvement in neurological scorecompared with vehicle treated rats (n=5).

Example 9 NRG1β Prevents Microglial and Astrocytic Activation FollowingMCAO

Rats were subjected to MCAO followed by reperfusion for 24 hours (n=5for each condition). NRG1β or vehicle was injected intraarterially asdescribed above. Sections were labeled for immunohistochemistry with anantibody against ED-1. As shown in FIGS. 10A-10F, while no staining wasseen in the contralateral side (FIG. 10A), ED-1 labeled cells arepresent in the ipsilateral hemisphere (FIG. 10B) following MCAO invehicle-treated animals. Few ED-1 positive cells are found in animalstreated with NRG1β (FIG. 10C). To assess astrocytic activation, sectionswere labeled for immunohistochemistry with an antibody against GFAP.Compared to the contralateral control (FIG. 10D), heavy GFAP staining isfound at the border or infarct (FIG. 10E) following MCAO invehicle-treated animals. However, when rats were treated with NRG-1β,GFAP expression was dramatically reduced in the peri-infarct regions(FIG. 10F).

Example 10 NRG1β Reduces MCAO/Reperfusion-Induced IL-1β mRNA Levels

Rats were treated with NRG1β or vehicle then subjected to MCAO. RNA wasisolated and IL1β mRNA expression was measured by RT-PCR. FIG. 11 showsthe mRNA expression levels of IL-1 (panel a) and GAPDH (panel b) (n=4for each condition). Panel c shows the average percentage of change±SEMin IL-1 mRNA levels from NRG1β-treated rat compared to vehicle-treatedcontrols after normalization to GAPDH.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

1-22. (canceled)
 23. A method for ameliorating neuronal damage in a subject, comprising: administering to said subject an effective amount of a pharmaceutical composition comprising: a chimeric neuregulin polypeptide having an integrin domain that binds to an integrin and an Erb3/4 binding domain that bind to Erb3 and/or Erb4, neuregulin polypeptide comprising: a neuregulin backbone derived from a native neuregulin polypeptide; and a donor fragment of at least one amino acid; wherein said donor fragment (1) replaces a target fragment in said native neuregulin polypeptide, wherein said donor fragment differs from said target fragment by at least one amino acid, or (2) is inserted into an insertion site of said neuregulin polypeptide; and wherein said donor fragment forms at least a portion of said integrin binding domain and/or at least a portion of said Erb3/4 binding domain of said chimeric neuregulin polypeptide, and a pharmaceutically acceptable carrier.
 24. The method of claim 23, wherein said neuronal damage is caused by an occlusive stroke and wherein said pharmaceutical composition is administered within 72 hours of said occlusive stroke.
 25. The method of claim 24, wherein said pharmaceutical composition is administered in conjunction with t-PA.
 26. The method of claim 24, wherein said pharmaceutical composition is administered in conjunction with a glutamate receptor inhibitor.
 27. The method of claim 24, wherein said pharmaceutical composition is administered after the initial inflammation due to the occlusive stroke has subsided to enhance migration of stem cells from a ventricle into an area of the brain damaged by the occlusive stroke.
 28. The method of claim 23, wherein said neuronal damage is caused by exposure to a neurotoxin and wherein said pharmaceutical composition is administered within 72 hours of said exposure.
 29. The method of claim 28, wherein said neurotoxin is an organophosphate.
 30. The method of claim 28, wherein the composition is administered in conjunction with an antidotes to neurotoxin or an anticonvulsant.
 31. The method of claim 23, wherein said neuronal damage is caused by acute CNS injuries, and wherein said composition is administered within 72 hours of the cause of CNS injury.
 32. The method of claim 23, wherein said neuronal damage is caused by a neurodegenerative disorder.
 33. The method of claim 32, wherein said neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease or amyotrophic lateral sclerosis.
 34. A method for preventing or ameliorating secondary neuronal injury and inflammation following traumatic brain injury (TBI), comprising: administering into a subject in need of such treatment an effective amount of a pharmaceutical composition comprising: a chimeric neuregulin polypeptide having an integrin domain that binds to an integrin and an Erb3/4 binding domain that binds to Erb3 and/or Erb4, said chimeric neuregulin polypeptide comprising: a neuregulin backbone derived from a native neuregulin polypeptide; and a donor fragment of at least one amino acid; wherein said donor fragment (1) replaces a target fragment in said native neuregulin polypeptide, wherein said donor fragment differs from said target fragment by at least one amino acid, or (2) is inserted into an insertion site of said neuregulin polypeptide; and wherein said donor fragment forms at least a portion of said integrin binding domain and/or at least a portion of said Erb3/4 binding domain of said chimeric neuregulin polypeptide, and a pharmaceutically acceptable carrier.
 35. A method for ameliorating blood vessel damage caused by acute mechanical or chemical assault in a subject, comprising: administering to said subject an effective amount of a pharmaceutical composition comprising: a chimeric neuregulin polypeptide having an integrin domain that binds to an integrin and an Erb3/4 binding domain that binds to Erb3 and/or Erb4, said chimeric neuregulin polypeptide comprising: a neuregulin backbone derived from a native neuregulin polypeptide; and a donor fragment of at least one amino acid; wherein said donor fragment (1) replaces a target fragment in said native neuregulin polypeptide, wherein said donor fragment differs from said target fragment by at least one amino acid, or (2) is inserted into an insertion site of said neuregulin polypeptide; and wherein said donor fragment forms at least a portion of said integrin binding domain and/or at least a portion of said Erb3/4 binding domain of said chimeric neuregulin polypeptide, and a pharmaceutically acceptable carrier.
 36. The method of claim 35, wherein said blood vessel damage is caused by placement of a balloon or stent in a blood vessel, diagnostic cardiac catheterization, or cardiac surgery. 